We propose that intraocular pressure (IOP) elevation in primary open angle glaucoma results from increased stiffness of Schlemm's canal (SC) endothelial cells, which impairs pore formation, and consequently obstructs aqueous humor outflow. This hypothesis is mechanistic, testable and when exploited can lead to novel therapies for glaucoma. Data obtained during the first award period of this BRP achieved all set milestones, and strongly supports the central hypothesis. We have shown that SC cells are highly contractile, modifying their contractile stresses and stiffnesses to levels comparable to smooth muscle cells. We have demonstrated that mechanical strain on SC cells potentiates pore formation. We have also discovered a remarkable link between cell stiffness and outflow resistance, specifically that drugs that increase (decrease) SC cell stiffness increase (decrease) resistance. Together, these observations demonstrate that SC cells are highly mechanosensitive and their biomechanical activity is tightly tied to aqueous outflow regulation and survival in a mechanically demanding environment. Moreover, our studies have further demonstrated that glaucomatous SC cells have altered mechanobiology including: (i) elevated cell stiffness, (ii) reduced pore-forming capability, and (iii) enhanced mechanosensitivity to substrate stiffness. This latter finding of ours is particularly relevant to recent findings of othrs, showing elevated stiffness in the trabecular meshwork of glaucomatous eyes. Our renewal application builds upon these milestones and focuses upon mechanism, genetics and translation to therapeutic applications. To test our hypothesis, we have designed five specific aims: First, we will extend a conceptual model we have developed that details the relationship between cell stiffness and outflow resistance. Second, we will look to extend our seminal findings of elevated stiffness of glaucomatous SC cells in vitro to the situation in situ, and will also examine whether the effect is magnified under physiological load. Third, we will determine the role of genetic regulatory processes in the altered biomechanics of glaucomatous SC cells, exploring underlying mechanisms. Fourth, we will introduce high-throughput functional (mechanobiologically based) screening to identify new candidate compounds that decrease SC cell contractile forces. Finally, we will use viral vectors to deliver cell stiffness-altering gene that specifically target the SC (and not TM, or other cell types) and monitor effects on outflow function. Testing of our hypothesis will enable rational development of targeted glaucoma therapies that selectively decrease cell stiffness at the level of Schlemm's canal, consequently reducing IOP.